The following tests are designed to determine the biological reactivity of mammalian cell cultures following contact with the elastomeric plastics and other polymeric materials with direct or indirect patient contact or of specific extracts prepared from the materials under test. It is essential that the tests be performed on the specified surface area. When the surface area of the specimen cannot be determined, use 0.1 g of elastomer or 0.2 g of plastic or other material for every mL of extraction fluid. Exercise care in the preparation of the materials to prevent contamination with microorganisms and other foreign matter.

Three tests are described (i.e., the Agar Diffusion Test, the Direct Contact Test, and the Elution Test).* The decision as to which type of test or the number of tests to be performed to assess the potential biological response of a specific sample or extract depends upon the material, the final product, and its intended use. Other factors that may also affect the suitability of sample for a specific use are the polymeric composition; processing and cleaning procedures; contacting media; inks; adhesives; absorption, adsorption, and permeability of preservatives; and conditions of storage. Evaluation of such factors should be made by appropriate additional specific tests before determining that a product made from a specific material is suitable for its intended use.

Autoclave
Employ an autoclave capable of maintaining a temperature of 121 ± 2, equipped with a thermometer, a pressure gauge, a vent cock, a rack adequate to accommodate the test containers above the water level, and a water cooling system that will allow for cooling of the test containers to about 20, but not below 20, immediately following the heating cycle.

Oven
Use an oven, preferably a mechanical convection model, that will maintain operating temperatures in the range of 50 to 70 within ± 2.

Incubator
Use an incubator capable of maintaining a temperature of 37 ± 1 and a humidified atmosphere of 5 ± 1% carbon dioxide in air.

Extraction Containers
Use only containers, such as ampuls or screw-cap culture test tubes, or their equivalent, of Type I glass. If used, culture test tubes, or their equivalent, are closed with a screw cap having a suitable elastomeric liner. The exposed surface of the elastomeric liner is completely protected with an inert solid disk 50 to 75 µm in thickness. A suitable disk can be fabricated from polytef.

Preparation of Apparatus
Cleanse all glassware thoroughly with chromic acid cleansing mixture and, if necessary, with hot nitric acid followed by prolonged rinsing with Sterile Water for Injection. Sterilize and dry by a suitable process containers and devices used for extraction, transfer, or administration of test material. If ethylene oxide is used as the sterilizing agent, allow not less than 48 hours for complete degassing.

Preparation of Extracts
Prepare as directed for Preparation of Extracts in Biological Reactivity Tests, In Vivo 88 using either Sodium Chloride Injection (0.9% NaCl) or serum-free mammalian cell culture media as Extraction Solvents. [NOTEIf extraction is done at 37 for 24 hours in an incubator, use cell culture media supplemented by serum. The extraction conditions should not in any instance cause physical changes, such as fusion or melting of the material pieces, other than a slight adherence.]

Agar Diffusion Test

This test is designed for elastomeric closures in a variety of shapes. The agar layer acts as a cushion to protect the cells from mechanical damage while allowing the diffusion of leachable chemicals from the polymeric specimens. Extracts of materials that are to be tested are applied to a piece of filter paper.

Sample Preparation
Use extracts prepared as directed or use portions of the test specimens having flat surfaces not less than 100 mm2 in surface area.

Positive Control Preparation
Proceed as directed for Sample Preparation.

Negative Control Preparation
Proceed as directed for Sample Preparation.

Procedure
Using 7 mL of cell suspension prepared as directed under Cell Culture Preparation, prepare the monolayers in plates having a 60-mm diameter. Following incubation, aspirate the culture medium from the monolayers, and replace it with serum-supplemented culture medium containing not more than 2% of agar. [NOTEThe quality of the agar must be adequate to support cell growth. The agar layer must be thin enough to permit diffusion of leached chemicals.] Place the flat surfaces of Sample Preparation, Negative Control Preparation, and Positive Control Preparation or their extracts in an appropriate extracting medium, in duplicate cultures in contact with the solidified agar surface. Use no more than three specimens per prepared plate. Incubate all cultures for not less than 24 hours at 37 ± 1, preferably in a humidified incubator containing 5 ± 1% of carbon dioxide. Examine each culture around each Sample, Negative Control, and Positive Control, under a microscope, using a suitable stain, if desired.

Interpretation of Results
The biological reactivity (cellular degeneration and malformation) is described and rated on a scale of 0 to 4 (see Table 1). Measure the responses of the cell cultures to the Sample Preparation, the Negative Control Preparation, and the Positive Control Preparation. The cell culture test system is suitable if the observed responses to the Negative Control Preparation is grade 0 (no reactivity) and to the Positive Control Preparation is at least grade 3 (moderate). The Sample meets the requirements of the test if the response to the Sample Preparation is not greater than grade 2 (mildly reactive). Repeat the procedure if the suitability of the system is not confirmed.

This test is designed for materials in a variety of shapes. The procedure allows for simultaneous extraction and testing of leachable chemicals from the specimen with a serum-supplemented medium. The procedure is not appropriate for very low- or high-density materials that could cause mechanical damage to the cells.

Sample Preparation
Use portions of the test specimen having flat surfaces not less than 100 mm2 in surface area.

Positive Control Preparation
Proceed as directed for Sample Preparation.

Negative Control Preparation
Proceed as directed for Sample Preparation.

Procedure
Using 2 mL of cell suspension prepared as directed under Cell Culture Preparation, prepare the monolayers in plates having a 35-mm diameter. Following incubation, aspirate the culture medium from the cultures, and replace it with 0.8 mL of fresh culture medium. Place a single Sample Preparation, a Negative Control Preparation, and a Positive Control Preparation in each of duplicate cultures. Incubate all cultures for not less than 24 hours at 37 ± 1 in a humidified incubator containing 5 ± 1% of carbon dioxide. Examine each culture around each Sample, Negative Control, and Positive Control Preparation, either visually or under a microscope, using a suitable stain, if desired.

Interpretation of Results
Proceed as directed for Interpretation of Results under Agar Diffusion Test. The Sample meets the requirements of the test if the response to the Sample Preparation is not greater than grade 2 (mildly reactive). Repeat the procedure if the suitability of the system is not confirmed.

Elution Test

This test is designed for the evaluation of extracts of polymeric materials. The procedure allows for extraction of the specimens at physiological or nonphysiological temperatures for varying time intervals. It is appropriate for high-density materials and for dose-response evaluations.

Sample Preparation
Prepare as directed in Preparation of Extracts, using either Sodium Chloride Injection (0.9% NaCl) or serum-free mammalian cell culture media as Extraction Solvents. If the size of the Sample cannot be readily measured, a mass of not less than 0.1 g of elastomeric material or 0.2 g of plastic or polymeric material per mL of extraction medium may be used. Alternatively, use serum-supplemented mammalian cell culture media as the extracting medium to simulate more closely physiological conditions. Prepare the extracts by heating for 24 hours in an incubator containing 5 ± 1% of carbon dioxide. Maintain the extraction temperature at 37 ± 1, because higher temperatures may cause denaturation of serum proteins.

Positive Control Preparation
Proceed as directed for Sample Preparation.

Negative Control Preparation
Proceed as directed for Sample Preparation.

Procedure
Using 2 mL of cell suspension prepared as directed under Cell Culture Preparation, prepare the monolayers in plates having a 35-mm diameter. Following incubation, aspirate the culture medium from the monolayers, and replace it with extracts of the Sample Preparation, Negative Control Preparation, or Positive Control Preparation. The serum-supplemented and serum-free cell culture media extracts are tested in duplicate without dilution (100%). The Sodium Chloride Injection extract is diluted with serum-supplemented cell culture medium and tested in duplicate at 25% extract concentration. Incubate all cultures for 48 hours at 37 ± 1 in a humidified incubator preferably containing 5 ± 1% of carbon dioxide. Examine each culture at 48 hours, under a microscope, using a suitable stain, if desired.

Interpretation of Results
Proceed as directed for Interpretation of Results under Agar Diffusion Test but using Table 2. The Sample meets the requirements of the test if the response to the Sample Preparation is not greater than grade 2 (mildly reactive). Repeat the procedure if the suitability of the system is not confirmed. For dose-response evaluations, repeat the procedure, using quantitative dilutions of the sample extract.

Table 2. Reactivity Grades for Elution Test

Grade

Reactivity

Conditions of all Cultures

0

None

Discrete intracytoplasmic granules; no cell lysis

1

Slight

Not more than 20% of the cells are round, loosely attached, and without intracytoplasmic granules; occasional lysed cells are present

2

Mild

Not more than 50% of the cells are round and devoid of intracytoplasmic granules; no extensive cell lysis and empty areas between cells

3

Moderate

Not more than 70% of the cell layers contain rounded cells or are lysed